The present invention relates to methods for determining the presence or absence of Mycoplasma pneumoniae in respiratory samples or other patient specimens or culture samples. The method involves using nucleic acid primers to amplify specifically a target sequence within the hmw gene cluster, preferably using one of the techniques of Strand Displacement Amplification (SDA), thermophilic Strand Displacement Amplification (tSDA) or fluorescent real time tSDA.
M. pneumoniae is predominantly a pathogen of the human respiratory tract and can cause bronchitis, pharyngitis and atypical pneumonia. It most commonly infects older children and young adults. Standard laboratory methods for diagnosis of M. pneumoniae include culture and serology. Both methods have disadvantages; M. pneumoniae is fastidious and requires 1 to 3 weeks to culture, while serology is insensitive and non-specific. Nucleic acid amplification methods for the detection of M. pneumoniae potentially offer the advantages of speed and improved sensitivity and specificity.
Physical mapping, as described by Wenzel, et al. (1988, Nucl. Acids Res. 16:8323-8336), and sequencing of the complete genome, as described by Himmelreich, et al. (1996, Nucl. Acids Res. 24:4420-4449), of M. pneumoniae has been performed. Several proteins believed to be involved in the attachment of this organism to host cells have been discovered. Protein products of the hmw gene cluster appear to play an accessory role in the adhesion of the M. pneumoniae organism to host epithelial cells (Baseman, et al., 1982, J. Bacteriol. 151:1514-1522; Krause, et al., 1982, Infect. Immun. 35:809-817; Krause, et al., 1983, Infect. Immun. 39:830-836; Stevens, et al., 1990, Infect. Immun. 58:3430-3433; Hahn, et al., 1998, J. Bacteriol. 180:1270-1276; Razin, et al., 1992, J. Gen. Microbiol. 138:407-422 and Krause, et al., 1996, Mol. Microbiol. 20:247-253). Sequence analysis (Dirksen, et al., 1996, Gene 171:19-25 and Ogle, et al., 1992, Infect. Immun. 60:1633-1641) and physical mapping (Krause, et al., 1991, Gene 107:83-89 and Stevens, et al., 1991, J. Bacteriol. 173:1041-1050) of the hmw gene cluster have been performed. Nucleic acid amplification is a powerful technology, which allows rapid detection of specific target sequences. It is therefore a promising technology for the rapid detection and identification of M pneumoniae. The oligonucleotide primers of the present invention are applicable to nucleic acid amplification and detection of M. pneumoniae. 
The following terms are defined herein as follows:
An amplification primer is a primer for amplification of a target sequence by extension of the primer after hybridization to the target sequence. Amplification primers are typically about 10-75 nucleotides in length, preferably about 15-50 nucleotides in length. The total length of an amplification primer for SDA is typically about 25-50 nucleotides. The 3xe2x80x2 end of an SDA amplification primer (the target binding sequence) hybridizes at the 3xe2x80x2 end of the target sequence. The target binding sequence is about 10-25 nucleotides in length and confers hybridization specificity on the amplification primer. The SDA amplification primer further comprises a recognition site for a restriction endonuclease 5xe2x80x2 to the target binding sequence. The recognition site is for a restriction endonuclease which will nick one strand of a DNA duplex when the recognition site is hemimodified, as described by G. Walker, et al. (1992, Proc. Natl. Acad. Sci. USA 89:392-396 and 1992, Nucl. Acids Res. 20:1691-1696). The nucleotides 5xe2x80x2 to the restriction endonuclease recognition site (the xe2x80x9ctailxe2x80x9d) function as a polymerase repriming site when the remainder of the amplification primer is nicked and displaced during SDA. The repriming function of the tail nucleotides sustains the SDA reaction and allows synthesis of multiple amplicons from a single target molecule. The tail is typically about 10-25 nucleotides in length. Its length and sequence are generally not critical and can be routinely selected and modified. As the target binding sequence is the portion of a primer which determines its target-specificity, for amplification methods which do not require specialized sequences at the ends of the target the amplification primer generally consists essentially of only the target binding sequence. For example, amplification of a target sequence according to the invention using the Polymerase Chain Reaction (PCR) will employ amplification primers consisting of the target binding sequences of the amplification primers described herein. For amplification methods that require specialized sequences appended to the target other than the nickable restriction endonuclease recognition site and the tail of SDA (e.g., an RNA polymerase promoter for Self-Sustained Sequence Replication (3SR), Nucleic Acid Sequence-Based Amplification (NASBA) or the Transcription-Based Amplification System (TAS)), the required specialized sequence may be linked to the target binding sequence using routine methods for preparation of oligonucleotides without altering the hybridization specificity of the primer.
A bumper primer or external primer is a primer used to displace primer extension products in isothermal amplification reactions. The bumper primer anneals to a target sequence upstream of the amplification primer such that extension of the bumper primer displaces the downstream amplification primer and its extension product.
The terms target or target sequence refer to nucleic acid sequences to be amplified. These include the original nucleic acid sequence to be amplified, the complementary second strand of the original nucleic acid sequence to be amplified and either strand of a copy of the original sequence which is produced by the amplification reaction. These copies serve as amplifiable targets by virtue of the fact that they contain copies of the sequence to which the amplification primers hybridize.
Copies of the target sequence that are generated during the amplification reaction are referred to as amplification products, amplimers or amplicons.
The term extension product refers to the copy of a target sequence produced by hybridization of a primer and extension of the primer by polymerase using the target sequence as a template.
The term species-specific refers to detection, amplification or oligonucleotide hybridization to a species of organism or a group of related species without substantial detection, amplification or oligonucleotide hybridization to other species of the same genus or species of a different genus.
The term assay probe refers to any oligonucleotide used to facilitate detection or identification of a nucleic acid. Detector probes, detector primers, capture probes, signal primers and reporter probes as described below are examples of assay probes.
A signal primer comprises a 3xe2x80x2 target binding sequence that hybridizes to a complementary sequence in the target and further comprises a 5xe2x80x2 tail sequence that is not complementary to the target (the adapter sequence). The adapter sequence is an indirectly detectable marker selected such that its complementary sequence will hybridize to the 3xe2x80x2 end of the reporter probe described below. The signal primer hybridizes to the target sequence at least partially downstream of the hybridization site of an amplification primer. The signal primer is extended by the polymerase in a manner similar to extension of the amplification primers. Extension of the amplification primer displaces the extension product of the signal primer in a target amplification-dependent manner, producing a single-stranded product comprising a 5xe2x80x2 adapter sequence, a downstream target binding sequence and a 3xe2x80x2 binding sequence specific for hybridization to a flanking SDA amplification primer. Hybridization and extension of this flanking amplification primer and its subsequent nicking and extension creates amplification products containing the complement of the adapter sequence which may be detected as an indication of target amplification.
A reporter probe according to the present invention functions as a detector oligonucleotide and comprises a label which is preferably at least one donor/quencher dye pair, i.e., a fluorescent donor dye and a quencher for the donor fluorophore. The label is linked to a sequence or structure in the reporter probe (the reporter moiety) which does not hybridize directly to the target sequence. The sequence of the reporter probe 3xe2x80x2 to the reporter moiety is selected to hybridize to the complement of the signal primer adapter sequence. In general, the 3xe2x80x2 end of the reporter probe does not contain sequences with any significant complementarity to the target sequence. If the amplification products containing the complement of the adapter sequence described above are present, they can then hybridize to the 3xe2x80x2 end of the reporter probe. Priming and extension from the 3xe2x80x2 end of the adapter complement sequence allows the formation of the reporter moiety complement. This formation renders the reporter moiety double-stranded, thereby allowing the label of the reporter probe to be detected and indicating the presence of or the amplification of the target.
The term amplicon refers to the product of the amplification reaction generated through the extension of either or both of a pair of amplification primers. An amplicon may contain exponentially amplified nucleic acids if both primers utilized hybridize to a target sequence. Alternatively, amplicons may be generated by linear amplification if one of the primers utilized does not hybridize to the target sequence. Thus, this term is used generically herein and does not imply the presence of exponentially amplified nucleic acids.
The present invention provides oligonucleotide primers that can be used for amplification of a target sequence found in M. pneumoniae. More specifically, the target sequence comprises a segment within the ORF9 region of the hmw gene cluster. The amplification primers have been designed for high-efficiency, high-specificity amplification at elevated temperatures, such as in tSDA and the PCR, however, they are also useful in lower-temperature amplification reactions such as conventional SDA, 3SR, TAS or NASBA. An oligonucleotide reporter probe that hybridizes to the complement of target specific signal primers is used to indirectly detect the amplification products.
The oligonucleotides of the invention may be used after culture as a means for confirming the identity of the cultured organism. Alternatively, they may be used for the detection and identification of M. pneumoniae in clinical samples from humans or animals using known amplification methods. In either case, the inventive oligonucleotides and assay methods provide a means for rapidly discriminating between M. pneumoniae and other microorganisms, allowing the practitioner to identify this microorganism rapidly without resorting to the more traditional procedures customarily relied upon. Such rapid identification of the specific etiological agent involved in an infection provides information that can be used to determine appropriate action within a short period of time.
SEQ ID NO: 1 is a sequence of an oligonucleotide used as an upstream primer for amplification of a sequence within the hmw gene cluster. SEQ ID NOs: 2-3 are sequences of oligonucleotides used as downstream primers for amplification of a sequence within the hmw gene cluster. SEQ ID NOs: 4-5 are sequences of oligonucleotides used as upstream bumpers for SDA amplification. SEQ ID NOs: 6-7 are sequences of oligonucleotides used as downstream bumpers for SDA amplification. SEQ ID NOs: 8-9 are sequences of signal primers for amplification and detection of a sequence within the hmw gene cluster. SEQ ID NO: 10 is a sequence for a reporter probe designed for detection of a sequence within the hmw gene cluster when used in conjunction with any of the aforementioned signal primers.